Physiological and behavioural implications of the portosystemic shunt in C57Bl/6J mice.

A significant fraction of the popular inbred C57Bl/6J mice show structural and biochemical features of the congenital portosystemic shunt (PSS). How this hepatic abnormality affects physiological and behavioural parameters has not been explored in detail. Here, we confirmed the frequent occurrence of the PSS in C57Bl/6J mice by three different methods. We screened a cohort of 119 C57Bl/6J mice for total bile acids (TBA) in plasma, identified 11 animals (9.2%) with high TBA (>11 µm; 171.1 ± 76.8 µm), and confirmed PSS presence in that subset by magnetic resonance angiography and 1H-magnetic resonance spectroscopy of brain metabolites in the hippocampal area. In addition to the high glutamine and low myo-inositol levels, we detected lower levels of several neurotransmitters and metabolites in the hippocampus, higher brain weight and volume, as well as enhanced brain glucose utilisation in the PSS mice. We also observed differences in peripheral organ weights, haematological cell counts and clinical chemistry parameters in C57Bl/6J mice with and without PSS. Animals with PSS were slightly hyperlocomotive, had better balance on the rotarod, showed altered gait properties, and displayed attenuated fear memory in the fear conditioning test. Furthermore, we revealed a significant alteration of the pharmacokinetic profile of diazepam in C57Bl/6J mice with PSS. Our data support previous reports of hepatic disturbances and demonstrate an altered neurobiological phenotype in C57Bl/6J mice with PSS. Such congenital differences between inbred C57Bl/6J littermates may significantly distort experimental outcomes of pharmacological, behavioural and genetic studies. KEY POINTS: A significant proportion of C57Bl/6J mice, an inbred strain popular in preclinical research, have congenital portosystemic shunts (PSS) that allow venous blood to enter systemic circulation bypassing the liver. In this study, we extended existing knowledge of PSS consequences, particularly with respect to the effects on brain structure and function. We demonstrated that C57Bl/6J mice with PSS differ from their normal counterparts in brain size and contents of several neuroactive substances, as well as in peripheral organ weights, rate of glucose utilisation, blood cell counts and blood clinical chemistry parameters. C57Bl/6J mice with PSS showed altered locomotor behaviour, performed worse in a memory test and had abnormal blood pharmacokinetics of a benzodiazepine drug after a single administration. PSS presence may significantly complicate the interpretation of experiments in C57Bl/6J mice; therefore, we propose that before their use in biomedical studies, these mice should be screened with a simple blood test.

An in vivo accelerated developmental myelination model for testing promyelinating therapeutics.

BACKGROUND: Therapeutic agents stimulating the process of myelination could be beneficial for the treatment of demyelinating diseases, such as multiple sclerosis. The efficient translation of compounds promoting myelination in vitro to efficacy in vivo is inherently time-consuming and expensive. Thyroid hormones accelerate the differentiation and maturation of oligodendrocytes, thereby promoting myelination. Systemic administration of the thyroid hormone thyroxine (T4) accelerates brain maturation, including myelination, during early postnatal development. The objective of this study was to validate an animal model for rapid testing of promyelinating therapeutic candidates for their effects on early postnatal development by using T4 as a reference compound. METHODS: Daily subcutaneous injections of T4 were given to Sprague Dawley rat pups from postnatal day (PND) 2 to PND10. Changes in white matter were determined at PND10 using diffusion tensor magnetic resonance imaging (DTI). Temporal changes in myelination from PND3 to PND11 were also assessed by quantifying myelin basic protein (MBP) expression levels in the brain using the resonance Raman spectroscopy/enzyme-linked immunosorbent assay (RRS-ELISA) and quantitative immunohistochemistry. RESULTS: DTI of white matter tracts showed significantly higher fractional anisotropy in the internal capsule of T4-treated rat pups. The distribution of total FA values in the forebrain was significantly shifted towards higher values in the T4-treated group, suggesting increased myelination. In vivo imaging data were supported by in vitro observations, as T4 administration significantly potentiated the developmental increase in MBP levels in brain lysates starting from PND8. MBP levels in the brain of animals that received treatment for 9 days correlated with the FA metric determined in the same pups in vivo a day earlier. Furthermore, accelerated developmental myelination following T4 administration was confirmed by immunohistochemical staining for MBP in coronal brain sections of treated rat pups. CONCLUSIONS: T4-treated rat pups had increased MBP expression levels and higher MRI fractional anisotropy values, both indications of accelerated myelination. This simple developmental myelination model affords a rapid test of promyelinating activity in vivo within several days, which could facilitate in vivo prescreening of candidate therapeutic compounds for developmental hypomyelinating diseases. Further research will be necessary to assess the utility of this platform for screening promyelination compounds in more complex demyelination disease models, such us multiple sclerosis.

Caspase-6 activity in a BACHD mouse modulates steady-state levels of mutant huntingtin protein but is not necessary for production of a 586 amino acid proteolytic fragment.

Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.

White matter degeneration with Unverricht-Lundborg progressive myoclonus epilepsy: a translational diffusion-tensor imaging study in patients and cystatin B-deficient mice.

PURPOSE: To study white matter (WM) changes in patients with Unverricht-Lundborg progressive myoclonus epilepsy (EPM1) caused by mutations in the cystatin B gene and in the cystatin B-deficient (Cstb-/-) mouse model and to validate imaging findings with histopathologic analysis of mice. MATERIALS AND METHODS: Informed consent was obtained and the study was approved by an institutional ethics committee. Animal work was approved by the Animal Experiment Board of Finland. Diffusion-tensor imaging and tract-based spatial statistics (TBSS) were used to compare fractional anisotropic (FA) results and axial, radial, and mean diffusion among patients with EPM1 (n = 19) and control subjects (n = 18). Ex vivo diffusion-tensor imaging and TBSS were used to compare Cstb-/- mice (n = 9) with wild controls (n = 4). Areas of FA decrease in mice were characterized by means of immunohistochemical analysis and transmission electron microscopy. Student t test statistics were applied to report significant findings (threshold-free cluster enhancement, P < .05). RESULTS: Patients with EPM1 showed significantly (P < .05) reduced FA and increased radial and mean diffusion in all major WM tracts compared with those of control subjects, shown as global FA decrease along the TBSS skeleton (0.41 ± 0.03 vs 0.45 ± 0.02, respectively; P < 5 × 10(-6)). Cstb-/- mice exhibited significantly reduced FA (P < .05) and antimyelin basic protein staining. Transmission electron microscopy revealed degenerating axons in Cstb-/- mice vs controls (979 axons counted, 51 degenerating axons; 2.09 ± 0.29 per field vs 1072 axons counted, nine degenerating axons; 0.48 ± 0.19 per field; P = .002). CONCLUSION: EPM1 is characterized by widespread alterations in subcortical WM, the thalamocortical system, and the cerebellum, which result in axonal degeneration and WM loss. These data suggest that motor disturbances and other symptoms in patients with EPM1 involve not only the cortical system but also the thalamocortical system and cerebellum.

Vertigoheel promotes rodent cognitive performance in multiple memory tests.

Introduction: Cognitive impairment associated with old age or various brain disorders may be very disabling for affected individuals, placing their carers and public health services under considerable stress. The standard-of-care drugs produce only transient improvement of cognitive impairment in older people, so the search for novel, safe and effective therapeutics that would help to reverse or delay cognitive impairment is warranted. Repurposing pharmacological therapies with well-established safety record for additional indications is a promising recent trend in drug development. Vertigoheel (VH-04), a multicomponent drug made of Ambra grisea, Anamirta cocculus L., Conium maculatum, and Petroleum rectificatum, has been successfully used for several decades in the treatment of vertigo. Here, we investigated effects of VH-04 on cognitive performance in standard behavioral tests assessing different types of memory and explored cellular and molecular underpinnings of VH-04's biological activity. Methods: In the majority of behavioral experiments, namely in the spontaneous and rewarded alternation tests, passive avoidance test, contextual/cued fear conditioning, and social transmission of food preference, we examined the ability of single and repeated intraperitoneal administrations of VH-04 to improve cognitive parameters of mice and rats disrupted by the application of the muscarinic antagonist scopolamine. In addition, we also assessed how VH-04 affected novel object recognition and influenced performance of aged animals in Morris water maze. Furthermore, we also studied the effects of VH-04 on primary hippocampal neurons in vitro and mRNA expression of synaptophysin in the hippocampus. Results: Administration of VH-04 positively influenced visual recognition memory in the novel object recognition test and alleviated the impairments in spatial working memory and olfactory memory caused by the muscarinic antagonist scopolamine in the spontaneous alternation and social transmission of food preference tests. In addition, VH-04 improved retention of the spatial orientation memory of old rats in the Morris water maze. In contrast, VH-04 did not have significant effects on scopolamine-induced impairments in tests of fear-aggravated memory or rewarded alternation. Experiments in vitro showed that VH-04 stimulated neurite growth and possibly reversed the age-dependent decrease in hippocampal synaptophysin mRNA expression, which implies that VH-04 may preserve synaptic integrity in the aging brain. Discussion: Our findings allow a cautious conclusion that in addition to its ability to alleviate manifestations of vertigo, VH-04 may be also used as a cognitive enhancer.

Cognitive disturbances in the cuprizone model of multiple sclerosis.

Cognitive problems frequently accompany neurological manifestations of multiple sclerosis (MS). However, during screening of preclinical candidates, assessments of behaviour in mouse models of MS typically focus on locomotor activity. In the present study, we analysed cognitive behaviour of 9 to 10-week-old female C57Bl/6J mice orally administered with the toxin cuprizone that induces demyelination, a characteristic feature of MS. Animals received 400 mg/kg cuprizone daily for 2 or 4 weeks, and their performance was compared with that of vehicle-treated mice. Cuprizone-treated animals showed multiple deficits in short touchscreen-based operant tasks: they responded more slowly to visual stimuli, rewards and made more errors in a simple rule-learning task. In contextual/cued fear conditioning experiments, cuprizone-treated mice showed significantly lower levels of contextual freezing than vehicle-treated mice. Diffusion tensor imaging showed treatment-dependent changes in fractional anisotropy as well as in axial and mean diffusivities in different white matter areas. Lower values of fractional anisotropy and axial diffusivity in cuprizone-treated mice indicated developing demyelination and/or axonal damage. Several diffusion tensor imaging measurements correlated with learning parameters. Our results show that translational touchscreen operant tests and fear conditioning paradigms can reliably detect cognitive consequences of cuprizone treatment. The suggested experimental approach enables screening novel MS drug candidates in longitudinal experiments for their ability to improve pathological changes in brain structure and reverse cognitive deficits.

A multimodal approach to identify clinically relevant biomarkers to comprehensively monitor disease progression in a mouse model of pediatric neurodegenerative disease.

While research has accelerated the development of new treatments for pediatric neurodegenerative disorders, the ability to demonstrate the long-term efficacy of these therapies has been hindered by the lack of convincing, noninvasive methods for tracking disease progression both in animal models and in human clinical trials. Here, we unveil a new translational platform for tracking disease progression in an animal model of a pediatric neurodegenerative disorder, CLN6-Batten disease. Instead of looking at a handful of parameters or a single "needle in a haystack", we embrace the idea that disease progression, in mice and patients alike, is a diverse phenomenon best characterized by a combination of relevant biomarkers. Thus, we employed a multi-modal quantitative approach where 144 parameters were longitudinally monitored to allow for individual variability. We use a range of noninvasive neuroimaging modalities and kinematic gait analysis, all methods that parallel those commonly used in the clinic, followed by a powerful statistical platform to identify key progressive anatomical and metabolic changes that correlate strongly with the progression of pathological and behavioral deficits. This innovative, highly sensitive platform can be used as a powerful tool for preclinical studies on neurodegenerative diseases, and provides proof-of-principle for use as a potentially translatable tool for clinicians in the future.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Systemic Delivery of Dysferlin Overlap Vectors Provides Long-Term Gene Expression and Functional Improvement for Dysferlinopathy.

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Hip region muscular dystrophy and emergence of motor deficits in dysferlin-deficient Bla/J mice.

The identification of a dysferlin-deficient animal model that accurately displays both the physiological and behavior aspects of human dysferlinopathy is critical for the evaluation of potential therapeutics. Disease progression in dysferlin-deficient mice is relatively mild, compared to the debilitating human disease which manifests in impairment of particular motor functions. Since there are no other known models of dysferlinopathy in other species, locomotor proficiency and muscular anatomy through MRI (both lower leg and hip region) were evaluated in dysferlin-deficient B6.A-Dysfprmd /GeneJ (Bla/J) mice to define disease parameters for therapeutic assessment. Despite the early and progressive gluteal muscle dystrophy and significant fatty acid accumulation, the emergence of significant motor function deficits was apparent at approximately 1 year of age for standard motor challenges including the rotarod, a marble bury test, grip strength, and swimming speed. Earlier observations of decreased performance for Bla/J mice were evident during extended monitoring of overall exploration and rearing activity. Comprehensive treadmill gait analyses of the Bla/J model indicated significant differences in paw placement angles and stance in relation to speed and platform slope. At 18 months of age, there was no significant difference in the life expectancy of Bla/J mice compared to wild type. Consistent with progressive volume loss and fatty acid accumulation in the hip region observed by MRI, mass measurement of individual muscles confirmed gluteal and psoas muscles were the only muscles demonstrating a significant decrease in muscle mass, which is analogous to hip-girdle weakness observed in human dysferlin-deficient patients. Collectively, this longitudinal analysis identifies consistent disease parameters that can be indicators of efficacy in studies developing treatments for human dysferlin deficiency.

Novel magnetic resonance imaging contrasts for monitoring response to gene therapy in rat glioma.

Magnetic resonance imaging relaxation times, T(1rho) and Carr-Purcell T(2) (CP-T(2)), were measured in a glioma herpes simplex virus-thymidine kinase gene therapy model. In treated tumors with >50% cell death by histology, T(1rho) and CP-T(2) measured with short spacing (tau(CP)) between centers of adiabatic refocusing pulses showed similar enhanced sensitivity to cytotoxic cell damage over CP-T(2) measured with long tau(CP) (long-tau(CP) T(2): 54.3 +/- 0.7 and 55.4 +/- 1.2 ms, P = 0.30; short-tau(CP) T(2): 61.3 +/- 1.0 and 64.2 +/- 1.1 ms, P < 0.05 before and day 2 of treatment, respectively). Without treatment, long-tau(CP) T(2) provided the most pronounced contrast between tumor and normal cerebral tissue. These data demonstrate that endogenous T(2) contrast can be modulated and extended in a manner likely to be clinically important.

Assignment of 1H nuclear magnetic resonance visible polyunsaturated fatty acids in BT4C gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death.

Polyunsaturated fatty acids (PUFAs), as detected by (1)H nuclear magnetic resonance (NMR) spectroscopy, accumulate into BT4C glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death (PCD). In this study, we have quantified the (1)H NMR visible lipids in vivo and characterized their biophysical and biochemical nature in these tumors during PCD both ex vivo and in vitro. Concentrations of (1)H NMR-detectable PUFAs increased 3-fold with pattern recognition identifying CH = CH and CH = CHCH(2)CH = CH as the most significant in monitoring the dynamics of PCD. The increase in PUFAs was equivalent to 70% of that in CH(2)CH(2)CH(2)-saturated lipid peak at 1.3 ppm. Ex vivo tumor samples, obtained from in situ funnel frozen tumors, showed very similar macromolecular peaks, as studied using high-resolution magic angle spinning (1)H NMR at 14.1 T, to those detected in vivo at 4.7 T. Line widths of lipid peaks were not influenced by the spin rate within the range of 1-9 kHz or temperature between 277 and 293 K, showing high degree of (1)H NMR detection of these peaks in vivo. These biophysical results additionally corroborate the idea that cytoplasmic lipid vesicles are the source of (1)H NMR lipid signals. Two-dimensional (1)H NMR ex vivo and tumor lipid extracts in vitro showed that the PUFA signals are in the same chemical compounds and consist of largely 18:1 and 18:2 lipids. Furthermore, it is suggested that the (1)H NMR lipids detected during PCD arise from cell constituent breakdown products forming lipid vesicles into dying cells.

Progressive volume loss and white matter degeneration in cstb-deficient mice: a diffusion tensor and longitudinal volumetry MRI study.

Unverricht-Lundborg type progressive myoclonus epilepsy (EPM1, OMIM 254800) is an autosomal recessive disorder characterized by onset at the age of 6 to 16 years, incapacitating stimulus-sensitive myoclonus and tonic-clonic epileptic seizures. It is caused by mutations in the gene encoding cystatin B. Previously, widespread white matter changes and atrophy has been detected both in adult EPM1 patients and in 6-month-old cystatin B-deficient mice, a mouse model for the EPM1 disease. In order to elucidate the spatiotemporal dynamics of the brain atrophy and white matter changes in EPM1, we conducted longitudinal in vivo magnetic resonance imaging and ex vivo diffusion tensor imaging accompanied with tract-based spatial statistics analysis to compare volumetric changes and fractional anisotropy in the brains of 1 to 6 months of age cystatin B-deficient and control mice. The results reveal progressive but non-uniform volume loss of the cystatin B-deficient mouse brains, indicating that different neuronal populations possess distinct sensitivity to the damage caused by cystatin B deficiency. The diffusion tensor imaging data reveal early and progressive white matter alterations in cystatin B-deficient mice affecting all major tracts. The results also indicate that the white matter damage in the cystatin B-deficient brain is most likely secondary to glial activation and neurodegenerative events rather than a primary result of CSTB deficiency. The data also show that diffusion tensor imaging combined with TBSS analysis provides a feasible approach not only to follow white matter damage in neurodegenerative mouse models but also to detect fractional anisotropy changes related to normal white matter maturation and reorganisation.

Diffusion tensor imaging and tract-based spatial statistics in Alzheimer's disease and mild cognitive impairment.

We aimed to explore the changes in fractional anisotropy (FA) in subjects with mild cognitive impairment (MCI) and Alzheimer's disease (AD) by analyzing diffusion tensor imaging (DTI) data using the Tract-Based Spatial Statistics (TBSS). DTI data were collected from 17 AD patients, 27 MCI subjects and 19 healthy controls. Voxel-based analysis with TBSS was used to compare FA among the three groups. Additionally, guided by TBSS findings, a region of interest (ROI)-based analysis along the TBSS skeleton was performed on group-level and the accuracy of the method was assessed by the back-projection of ROIs to the native space FA. Neurofiber tracts with decreased FA included: the parahippocampal white matter, cingulum, uncinate fasciculus, inferior and superior longitudinal fasciculus, corpus callosum, fornix, tracts in brain stem, and cerebellar tracts. Quantitative ROI-analysis further demonstrated the significant decrease on FA values in AD patients relative to controls whereas FA values of MCI patients were found in between the controls and AD patients. We conclude that TBSS is a promising method in examining the degeneration of neurofiber tracts in MCI and AD patients.

Stem cell profiling by nuclear magnetic resonance spectroscopy.

The classification of embryonic and adult stem cells, including their derivatives, is still limited, and often these cells are best defined by their functional properties. Recent gene array studies have yielded contradictory results. Also, very little is known about the metabolic properties of these exciting cells. In this study, proton (1H) NMR spectroscopy was used to identify the major low-molecular-weight metabolites in murine embryonic stem cells (ESC) and their neural stem cell (NSC) derivatives. ESC are characterized by an unusually low number of NMR-detectable metabolites, high phosphocholine (PC) content, and nondetectable glycerophosphocholine (GPC). The metabolic profiles of NSC resemble glial cells and oligodendrocyte progenitors, but with considerably higher PC, GPC, and myo-inositol content. The results suggest that NMR spectroscopy in vitro can provide markers to study the effects of differentiation on cell metabolism, and potentially to assess stem cell preparations for differentiation status.

High-resolution magic-angle-spinning 1H NMR spectroscopy reveals different responses in choline-containing metabolites upon gene therapy-induced programmed cell death in rat brain glioma.

Changes in the concentrations of choline-containing metabolites (CCM) have been implicated in both cell proliferation and death processes. In this study, high-resolution magic-angle-spinning (HRMAS) 1H NMR spectroscopy was used to study metabolite changes in the CCM chemical shift region in rat glioma ex vivo during apoptosis induced by thymidine kinase-ganciclovir gene therapy. Cell density and apoptotic activity in the tumours were quantified by histological methods. HRMAS 1H NMR was able to resolve peaks from choline (Cho), glycerophosphocholine (GPC), phosphocholine (PC), taurine (Tau) and myo-inositol (myo-Ins), all of which contribute to the in vivo 1H NMR peak centred at 3.23 ppm. The early phase of apoptosis (treatment day 4), with a approximately 2.8-fold increase in the number of apoptotic nuclei (at constant cell density of 1.8 +/- 0.1 x 10(5) cells/mm3) was associated with increases in resonance intensity from GPC and PC, while Cho and Tau remained unchanged. Later stage apoptosis, accompanied by synchronous cell death (cell density declined to 0.7 +/- 0.02 x 10(5) cells/mm3), resulted in a significant decline in Tau relative to untreated tumours, while the contents of CCMs and myo-Ins detectable by 1H HRMAS were unchanged. These observations demonstrate that, while the in vivo 1H NMR peak at 3.23 ppm is indicative of cellular processes involved in apoptosis, the biochemical changes monitored by this resonance involve a number of different and chemically distinct metabolites.

Water diffusion in a rat glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death in vivo: correlation with cell density.

PURPOSE: To study the characteristics of diffusion magnetic resonance imaging (MRI) contrast in a rat brain BT4C glioma during progression of ganciclovir (GCV)-thymidine kinase gene therapy-induced programmed cell death (PCD) in vivo. MATERIALS AND METHODS: The trace of the diffusion tensor (Dav = 1/3TraceD), T2, and spin density were determined by MRI and the apparent diffusion coefficient (ADC) of water by diffusion nuclear MR (NMR) spectroscopy using largely varying b values and diffusion times (tD) at 4.7 T. Cell count and apoptotic cells were quantified by histological means. RESULTS: Decline in cell count was strongly associated with increase in both Dav and T2. Spin density ratio between tumor and contralateral parietal cortex increased with a very similar time course as Dav and T2, indicating net water gain into the eradicating tumor. Diffusion spectroscopy showed a nonmonoexponential signal decay at all tD values ranging from 14-192 msec. During PCD, the ADC of the component yielding fast diffusion coefficient (D1), as acquired with tD > or = 47 msec, increased with kinetics similar to those of Dav (tD = 4.8 msec). The fractional size of D1 increased by 10% to 15% throughout the entire tD range. Apparent water residence time of the slow diffusion component, D2, shortened from a value of 38.3 +/- 1.7 msec on day 0 to 33.4 +/- 0.5 msec by day 8. CONCLUSION: The present results show that reduced cell density and increased water content, leading to altered water microenvironment, are associated with increased water diffusion coefficient in eradicating gliomas as a result of PCD.

Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo, ex vivo, and in vitro.

Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1H NMR visible lipids increased. HRMAS 1H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1H and 31P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo.